CN103820581B - A kind of test kit and method detecting human parvovirus B19 - Google Patents
A kind of test kit and method detecting human parvovirus B19 Download PDFInfo
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Abstract
The invention discloses a kind of test kit detecting human parvovirus B19, does it comprise nucleotide sequence as SEQ? ID? primer pair shown in NO:1 ~ 2, increases from the gene of human parvovirus B19.Test kit provided by the invention can increase the gene fragment of human parvovirus B19, and high specificity is highly sensitive, reproducible, for rapid detection blood products, has a good application prospect.
Description
Technical field
The present invention relates to a kind of test kit detecting human parvovirus B19, belong to biological technical field.
Background technology
Human parvovirus B19 (HumanparvovirusB19) be a kind of diameter be about 18 ~ 26nm without coating single-stranded DNA viruses, belong to the red Tobamovirus of Parvoviridae, erythema infectiosum, acute arthritis can be caused, infect pregnant woman and can cause fetus edema and even foetal death.General by respiratory infectious, also can propagate through blood transfusion, blood products and blood-tire approach.Whether the diameter of human parvovirus B19 is little and without coating, deactivation and removal job very difficult, therefore, need the detection method setting up a kind of B19, effectively detect containing B19 in blood products, to improve the security of blood products.
At present, the detection method of B19 is divided into the large technology of serology, molecular biology and histology three.Wherein, take ELISA as the Serological testing of representative and be that the molecular Biological Detection of representative is comparatively easy with PCR.But due to B19 virus not easily vitro culture, antigen preparation is also more difficult, causes the Serologic detection of B19 to be difficult to generally apply.PCR detects then not to be needed to cultivate virus, does not need to prepare antigen yet, more easily promotes the use of.
Zhao Guoqiang etc., " foundation of FQ-PCR method for detection of B 19 virus ", the documents such as Zhengzhou University's journal (medicine) volume the 3rd phase May the 40th in 2005 disclose the method adopting QPCR specific detection B19 virus, but, these methods all need to adopt probe to detect, to guarantee specificity and the accuracy of detection method, cause testing cost too high.
Need to find the more cheap PCR detection method of a kind of cost.
Summary of the invention
For solving the problem, the invention provides test kit and method that a kind of new PCR detects human parvovirus B19.
The present invention detects the test kit of human parvovirus B19, comprises the primer pair of nucleotide sequence as shown in SEQIDNO:1 ~ 2, increases from the gene of human parvovirus B19.
Described test kit also comprises positive control template: the DNA molecular containing nucleotide sequence gene fragment as shown in SEQIDNO:3.
Preferably, described DNA molecular is the recombinant plasmid comprising nucleotide sequence shown in SEQIDNO:3.Further preferably, described recombinant plasmid is restructuring PMD19-T plasmid.
Present invention also offers a kind of method detecting human parvovirus B19, it comprises the steps:
(1) Template preparation: extract the DNA in sample to be checked;
(2) increase: adopt the primer pair of nucleotide sequence as shown in SEQIDNO:1 ~ 2, the sample DNA extracted with step (1) for template, pcr amplification.
(3) result detects: detect amplification.
Wherein, in step (1), described sample to be checked is blood products.
Wherein, in step (2), described pcr amplification with the DNA molecular containing nucleotide sequence gene fragment as shown in SEQIDNO:3 for positive control template.
Preferably, described DNA molecular is the recombinant plasmid comprising nucleotide sequence shown in SEQIDNO:3.Further preferably, described recombinant plasmid is restructuring PMD19-T plasmid.
Wherein, described PCR is quantitative fluorescent PCR.
Quantitative fluorescent PCR, refers to and add fluorescence conjugate matter or fluorescently-labeled probe in PCR reaction system, utilizes fluorescent signal to accumulate the whole PCR process of Real-Time Monitoring, finally by typical curve, unknown template is carried out to the method for quantitative analysis.
Invention further provides the purposes of nucleotide sequence shown in SEQIDNO:1 ~ 2, it is for increasing or detecting the gene from human parvovirus B19.
The present invention specifically detects primer, the gene of specific amplified B19 when not using probe by design, and meanwhile, highly sensitive, reproducible, accuracy is high, effectively can detect the content of B19 virus in blood products, have a good application prospect.
The embodiment of form by the following examples, does foregoing of the present invention and describes in detail further.But the scope that should not be construed as the above-mentioned theme of the present invention is only limitted to following embodiment.All technology realized based on the content of claims of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
The agarose gel electrophoresis qualification of Fig. 1 PCR primer.1:DNAmaker, 100bp increase progressively; 2:B19DNA amplified production; 3: recombinant plasmid amplified production; 4: negative control
Fig. 2 specific amplification detection curve
The melting curve of Fig. 3 QPCR;
The typical curve of Fig. 4 QPCR;
Dependency between Fig. 5 sample detection result and actual value.
Embodiment
Test materials:
1. virus strain
The positive blood plasma (preservation of Xue Yuan inspection center of Rongsheng Pharmaceutical Co., Ltd., Chengdu) of human parvovirus B19; Pig parvoviral PPV(Porcineparvovirus, ATCC, lot number: 2547326); Pseudorabies virus PRV(Pseudorabiesvirus, Chinese animal doctor supervises institute, lot number: 198777); Bovine parvovirus BPV(Bovineparvovirus, Chengdu Inst. of Biological Products, lot number: 20111118); Mouse parvovirus MVM(Minutevirusofmice, ATCC, lot number: 58073406).
2. main agents and articles for use
High purity Viral nucleic acid extraction reagent box (HighPureViralNucleicKit, Roche, 11858874001); Plasmid reclaims test kit (Takara); Gammavenin (IVIG, Rongsheng Pharmaceutical Co., Ltd., Chengdu); L, P and S tri-kinds of 20nm filters purchased from three different manufacturers, are conventional commercial product respectively.
The design of embodiment 1 primer and the preparation of positive template
1. method
The Design and synthesis of 1.1 primers
According to the sequence (NC_000883) of B19 virus on NCBI, the VP1 district of B19 designs primer, upstream primer (P1) is 5'-GGCACCTCTCAAAACACT-3'(SEQIDNO:1); Downstream primer (P2) is 5'-CCACTCCTTGCTGATACTC-3'(SEQIDNO:2).Entrust the synthesis of Takara company.
1.2 adopt the primer amplification B19 gene designed
1.2.1 nucleic acid extraction
Extract the positive blood plasma of B19 and the nucleic acid containing the positive blood plasma IVIG of B19 with HighPureViralNucleicKit test kit, using the nucleic acid of the B19 negative plasma of test kit extraction as negative control, preserve under-40 DEG C of conditions.
1.2.2 amplification and qualification
With the genomic dna of B19 for template, with P1 and P2 for primer, pcr amplification:
Reaction system (cumulative volume 25 μ l): reaction premixed liquid (
, article No. R040A, Takara company) 12.5 μ l, primer 1 μ l(100pmol), sample DNA templates 1 μ l(1 μ g), with ddH
2o complements to 25 μ l.
Reaction conditions: 95 DEG C of sex change 5min; 94 DEG C of 1min, 52 DEG C of 1min, 72 DEG C of 45s, rear 3 steps carry out 30 circulations; 4 DEG C of preservations.
Electroresis appraisal is carried out with 1% sepharose.
The structure of 1.3 positive control template (standard substance) (recombinant plasmid PMD19-T-VP1) and qualification
After step 1.2 electroresis appraisal; glue reclaims the fragment increasing and obtain; be connected with PMD19-T carrier, obtain recombinant plasmid, transform Top10 competence bacteria; Invitrogen is sent to check order positive plasmid bacterial strain correct for bacterium colony PCR preliminary evaluation; and reclaim test kit with plasmid and reclaim, obtain PMD19-T-VP1 recombinant plasmid, with ultraviolet spectrophotometer measure under the condition of 260nm and 280nm respectively reclaim purity and the concentration of plasmid; after being diluted to 108copies/ μ l ,-40 DEG C save backup.
With the PMD19-T-VP1 recombinant plasmid built for template, with P1 and P2 for primer, pcr amplification, the reaction system of amplification and reaction conditions are with aforementioned 1.2.2.
Electroresis appraisal is carried out with 1% sepharose.
2, detected result
Experimental result as shown in Figure 1, adopts primer amplification B19 gene of the present invention, and obtaining length is 160bp(swimming lane 2) amplified fragments, its sequence is as follows:
SEQIDNO:3：
5’-GGCACCTCTCAAAACACTAGAATATCCTTACGCCCTGGGCCAGTGTCTCAGCCATATCACCACTGGGACACAGATAAATATGTCACAGGTATAAATGCCATTTCTCATGGTCAAACCACATATGGTAATGCTGAAGATAAAGAGTATCAGCAAGGAGTGG-3’
To comprise the recombinant plasmid PMD19-T-VP1 of aforementioned amplified fragments for template, also uniquely can increase and obtain the gene fragment (swimming lane 3) that length is 160bp, illustrate that the recombinant plasmid PMD19-T-VP1 prepared can use as positive template (standard substance).
Experimental result illustrates, the primer of the present invention's design can effectively increase the gene of B19.
The specific detection of embodiment 2 primer of the present invention
1, method
The DNA of B19, PRV, BPV, MMV, PK-15 is extracted as template respectively by the method for 1.2.1 in embodiment 1, pcr amplification, each sample arranges three multiple holes, B19(hole 1, hole 2, hole 3), PRV(hole 4, hole 5, hole 6), BPV(hole 7, hole 8, hole 9), MMV(hole 10, hole 11, hole 12), PK-15(hole 13, hole 14, hole 15):
Reaction system (cumulative volume is 15 μ l): EvaGreen premixed liquid (SsoFast
tM supermix, 172-5201, Bio-rad company) 7.5 μ l, each 0.5 μ l of upstream and downstream primer, template DNA 1 μ l(be with ddH
2o supplies 15 μ l).
Reaction conditions: 95 DEG C of 5min; 95 DEG C of 10s, 52 DEG C of 10s and 72 DEG C 10s, rear 3 steps repeat 40 circulations.
Check amplification curve.
2, result
As shown in Figure 2, each circulating collection fluorescent signal in PCR process, along with the increase of reaction cycle number of times, the sample in 1 ~ hole, hole 3, fluorescence intensity strengthens, and in level and smooth index curve, illustrates that the sample template in 1 ~ hole, hole 3 has carried out effective amplification; And the sample in hole 4 ~ 15, in whole reaction process, can't detect the change of fluorescent signal, illustrate that the sample in hole 4 ~ 15 does not increase.
Therefore, primer of the present invention can increase B19 gene (1 ~ hole, hole 3), but the gene of can not increase PRV, BPV, MMV, PK-15 (hole 4 ~ 15).
Experimental result illustrates, primer of the present invention can the gene of specific amplified B19, and genes of can not increase other viruses or cell, high specificity.
The sensitivity technique of embodiment 3 primer of the present invention
1, method
Positive for B19 high density blood plasma according to the form below 1 is diluted, the sample of the B19 of different titers must be contained;
Extract sample nucleic respectively as template, pcr amplification by the method for 1.2.1 in embodiment 1, repeat 3 times in the different time, each each concentration makees 8 parallel pipes:
Reaction system (cumulative volume is 15 μ l): each 0.5 μ l of EvaGreen premixed liquid 7.5 μ l, upstream and downstream primer, template DNA 1 μ l(supply 15 μ l with ddH2O).
Reaction conditions: 95 DEG C of 5min; 95 DEG C of 10s, 52 DEG C of 10s and 72 DEG C 10s, rear 3 steps repeat 40 circulations.
Check amplification curve, detect if having amplified reaction curve to count in 40 CP values, do not detect if counted without amplified reaction curve in 40 CP values.
2, result
Sensitivity experiments the results are shown in Table 1:
Table 1QPCR method detects the susceptibility of B19
As shown in Table 1, when the titre of B19 in testing sample is 10
3copies/ μ l, 10
2during copies/ μ l and 50copies/ μ l, the positive rate of sample is 100%; When the titre of B19 in testing sample is 10copies/ μ l, the positive rate of sample is 79%.
When the titre of B19 in measuring samples is low to moderate 50copies/ μ l, still can 100% accurately to detect, when the titre of B19 in measuring samples is low to moderate 10copies/ μ l, recall rate is also up to 79%.
Experimental result illustrates, primer of the present invention highly sensitive.
The accuracy of embodiment 4 primer of the present invention detects
1, method
The acquisition of 1.1 viruses and dilution
(in blood plasma, the titre of B19 is 2.07 × 10 to get the positive blood plasma of B19 high density
9copies/ μ l), be diluted to 7 gradients, after dilution, in blood plasma, the titre of B19 is as shown in the table:
The positive blood plasma dilution scheme of table 2
| Sample number into spectrum | Sample titre (copies/ul) |
| 1 | 10 8 |
| 2 | 10 7 |
| 3 | 10 6 |
| 4 | 10 5 |
| 5 | 10 4 |
| 6 | 10 3 |
| 7 | 10 2 |
1.2PCR amplification
The nucleic acid of the positive blood plasma of B19 of different titers is extracted as sample template by the method for 1.2.1 in embodiment 1, with the PMD19-T-VP1 recombinant plasmid of different concns for positive template, fluorescence real-time quantitative PCR increases, and repeat 3 times in the different time, each each concentration makees 8 parallel pipes.
Reaction system (cumulative volume is 15 μ l): each 0.5 μ l of EvaGreen premixed liquid 7.5 μ l, upstream and downstream primer, template DNA 1 μ l(supply 15 μ l with ddH2O).
Reaction conditions: 95 DEG C of 5min; 95 DEG C of 10s, 52 DEG C of 10s and 72 DEG C 10s, rear 3 steps repeat 40 circulations.
1.3 detect
After pcr amplification terminates, carry out melting curve analysis immediately by pre-set program: final step is warming up to 95 DEG C after having increased, be then cooled to 60 DEG C, maintain 20s, be warming up to 95 DEG C with 0.1 DEG C/s again, monitor the fluorescent value be warming up in 95 DEG C of time periods from 60 DEG C, make melting curve.
With PMD19-T-VP1 recombinant plasmid detected result, build typical curve, according to typical curve, determine the titre of different sample, contrast with the actual titre of sample, do X-coordinate with the logarithmic value of the actual titre of sample, do ordinate zou mapping with the detection titre of sample, calculate relation conefficient.
2, result
As seen from Figure 3, melting curve only has 1 peak, illustrates that primer amplification of the present invention obtains 1 amplified production, consistent with the amplification of embodiment 1, further illustrates primer of the present invention and can effectively to increase B19 gene.
As shown in Figure 4, the titre of the different samples adopting the inventive method to detect and the contrast of the actual titre of sample are as shown in Figure 5 for typical curve.As seen from Figure 5, the relation conefficient between the titre adopting the inventive method to detect and the actual titre of sample is 0.9989, illustrates that the inventive method accurately can detect the content of B19 in measuring samples.
Experimental result illustrates, the accuracy adopting primer of the present invention to detect is high.
The repeatability of embodiment 5 primer of the present invention detects
1, method
The acquisition of 1.1 viruses and dilution
With embodiment 4.
1.2PCR amplification
Extract the nucleic acid of the positive blood plasma of B19 of different titers by the method for 1.2.1 in embodiment 1 as sample template, fluorescence real-time quantitative PCR increases, and repeat 3 times in the different time, each each concentration makees 8 parallel pipes.
Calculate in test and the variation coefficient CV% of test bay Ct, detect the method repeatability.
2, result
Repeated experiment the results are shown in Table 3:
The Ct value result of table 3 different concns sample continuous detecting,
As shown in Table 3, in experimental group, CV% value maximum value is 3.89%, and mean value is 0.72%, and between group, CV% value maximum value is 2.65%, show present method batch in and batch between repeatability all better.
Experimental result illustrates, adopt primer of the present invention to detect, reproducible, numerical value is accurately credible.
The composition of embodiment 6 test kit of the present invention and using method
1, the composition of test kit
1.1 primers: 200 μ l, containing, for example the primer pair of nucleotide sequence shown in SEQNO:1 ~ 2, upstream and downstream primer concentration is 10nmol/ml.
1.2 reaction premixed liquids: 500 μ l × 5, purchased from Takara company, commodity are called PrimeSTAR zero RHS, and article No. is R040A, wherein containing archaeal dna polymerase, dNTPs, damping fluid.
1.3 positive controls: 200 μ l, prepare containing, for example nucleotide sequence positive plasmid PMD19-T-VP1(embodiment 1 of gene fragment as shown in SEQNO:3), the copy number of contained plasmid is 10
8copies/ μ l.
Above composition all needs-20 DEG C of preservations, takes out and melts, of short duration centrifugal rear use during use.
2, the using method (Standard PCR reaction system) of test kit
Concrete use step is:
(1) template extraction: the DNA extracting sample to be tested with nucleic acid extraction kit (e.g., HighPureViralNucleicKit test kit);
(2) increase: with the sample DNA of step (1) for template, adopt the primer in aforementioned agents box to carry out pcr amplification; Reaction system (25 μ l): react premixed liquid 12.5 μ l, primer 1 μ l, sample DNA templates or positive control template 1 μ l, complement to 25 μ l with ddH2O; Reaction conditions: 95 DEG C of 5min, 94 DEG C of 1min, 52 DEG C of 1min, 72 DEG C of 45S, rear 3 steps carry out 30 circulations.
(3) detect: amplified production 1% agarose gel electrophoresis analysis.
The composition of embodiment 7 test kit of the present invention and using method
1, the composition of test kit
1.1 primers: 250 μ l, containing, for example the primer pair of nucleotide sequence shown in SEQNO:1 ~ 2, upstream and downstream primer concentration is 10nmol/ml;
1.2 reaction premixed liquid: 5ml, purchased from Bio-rad company, commodity are called SsoFast
tM supermix, article No. is: 172-5201, wherein merges polysaccharase containing dNTPs, Sso7d, MgCl
2, EvaGreen dyestuff;
1.3 standard substance (positive control template): 250 μ l, prepare containing, for example nucleotide sequence positive plasmid PMD19-T-VP1(embodiment 1 of gene fragment as shown in SEQNO:3), the copy number of contained plasmid is 10
9copies/ μ l.
Above composition all needs-20 DEG C of preservations, takes out and melts, of short duration centrifugal rear use during use.
2, the using method of test kit
Concrete use step is:
(1) template extraction: the DNA extracting sample to be tested with nucleic acid extraction kit (e.g., HighPureViralNucleicKit test kit);
(2) increase: respectively with the standard substance of the sample DNA of step (1) and concentration gradient dilution for template, adopt the primer in aforementioned agents box to carry out fluorescence real-time quantitative PCR amplification; Reaction system 15ul: react premixed liquid 7.5ul, each 0.5ul of upstream and downstream primer, DNA profiling 1ul, supply 15ul with ddH2O; Reaction conditions: 95 DEG C of 5min, 95 DEG C of 10s, 52 DEG C of 10s, 72 DEG C of 10s, rear 3 steps carry out 40 circulations.
(3) detect: build typical curve, determine the content of B19 in measuring samples.
Embodiment 8 adopts test kit of the present invention to detect blood plasma product
1. method
1.1 preparation of samples
Primitive plasma: containing the positive blood plasma of B19;
Diluting plasma: by 1ml primitive plasma with 99mlIVIG(without B19) mix, to obtain final product;
0.1 μm of pre-filtering blood plasma: by diluting plasma 0.1 μm of frit, to obtain final product;
20nm filtered plasma: by 0.1 μm of pre-filtering blood plasma 20nm frit, to obtain final product.
1.2QPCR(fluorescence real-time quantitative PCR) detect
Using above-mentioned four kinds of blood plasma as sample to be checked, adopt the test kit of embodiment 7, detect according to the method for embodiment 7, wherein, the concentration of positive recombinant plasmid (standard substance) is: 10
7copies/ μ l arrives
10
2copies/μl。
2, result
From theory:
Diluting plasma dilutes 100 times on the basis of primitive plasma, and in diluting plasma, the content of B19 should be 1/100 of B19 content in primitive plasma;
0.1 μm of pre-filtering can remove virus, but poor removal effect, and thus in 0.1 μm of pre-filtering blood plasma, the content of B19 should be lower than the content of B19 in diluting plasma, but the two gap is not too large;
It is virus removing method general at present that 20nm filters, and its virus removal is effective, and thus in 20nm filtered plasma, the content of B19 should well below the content of B19 in 0.1 μm of pre-filtering blood plasma.
Actual detected result is as shown in table 4:
The content of B19 in table 4 four kinds of samples
| Sample | Primitive plasma | Diluting plasma | 0.1um pre-filtering blood plasma | 20nm filtered plasma |
| B19 content (copies/ul) | 2×10 9 | 2.1×10 7 | 1.7×10 7 | 7.98×10 3 |
As shown in table 4:
The B19 content of diluting plasma is about 1/100 of primitive plasma;
In 0.1 μm of pre-filtering blood plasma, the content of B19 is lower than the content of B19 in diluting plasma, but the order of magnitude of the two is identical, and gap is little;
The content of B19 four orders of magnitude lower than 0.1 μm of pre-filtering blood plasma in 20nm filtered plasma, the two gap clearly.
Can find out according to above-mentioned detected result, adopt test kit of the present invention to detect the content of B19 in sample, detected result is consistent with notional result, illustrates that test kit of the present invention effectively can detect the content of B19 in sample.
To sum up, test kit of the present invention is used for the detection of human parvovirus B19, and specificity is good, and susceptibility is high, and accuracy is high, reproducible, easy and simple to handle, effectively can detect the content of B19 in blood products, have a good application prospect.
Claims (4)
1. detect a human parvovirus B19's test kit, it is characterized in that: comprise the primer pair of nucleotide sequence as shown in SEQIDNO:1 ~ 2, increase from the gene of human parvovirus B19.
2. test kit according to claim 1, is characterized in that: described test kit also comprises positive control template: the DNA molecular containing nucleotide sequence gene fragment as shown in SEQIDNO:3.
3. test kit according to claim 2, is characterized in that: described DNA molecular is the recombinant plasmid containing nucleotide sequence gene fragment as shown in SEQIDNO:3.
4. test kit according to claim 3, is characterized in that: described recombinant plasmid is restructuring PMD19-T plasmid.
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| CN201410098048.0A CN103820581B (en) | 2014-03-17 | 2014-03-17 | A kind of test kit and method detecting human parvovirus B19 |
| PCT/CN2014/074269 WO2015139334A1 (en) | 2014-03-17 | 2014-03-28 | Kit and method for detecting human parvoviridae b19 |
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| CN107058629A (en) * | 2017-05-04 | 2017-08-18 | 张家港蓝苏生物工程有限公司 | A kind of fluorescence PCR method and kit of specific detection Human parvovirus B19 nucleic acid |
| CN110951921A (en) * | 2019-12-27 | 2020-04-03 | 苏州药明检测检验有限责任公司 | Primer, probe, kit and method for detecting human parvovirus B19 based on real-time fluorescent PCR technology |
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| CN1636071A (en) * | 2001-06-28 | 2005-07-06 | 希龙公司 | Diagnostic assays for parvovirus B19 |
| CN103451320A (en) * | 2013-08-09 | 2013-12-18 | 中国人民解放军军事医学科学院野战输血研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for three genotypes of human parvovirus B19, as well as universal detection primer, TaqMan probe and kit thereof |
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- 2014-03-28 WO PCT/CN2014/074269 patent/WO2015139334A1/en active Application Filing
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| CN1636071A (en) * | 2001-06-28 | 2005-07-06 | 希龙公司 | Diagnostic assays for parvovirus B19 |
| CN103451320A (en) * | 2013-08-09 | 2013-12-18 | 中国人民解放军军事医学科学院野战输血研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for three genotypes of human parvovirus B19, as well as universal detection primer, TaqMan probe and kit thereof |
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| WO2015139334A1 (en) | 2015-09-24 |
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